In eukaryotes, two distinct mechanisms have evolved in cells to initiate translation. In one of them, the methyl-7-guanosine (5′)pppN structure present at the 5′ end of the mRNA (the “cap”) is recognized by the initiation factor eIF4F, which is composed of eIF4E, eIF4G and eIF4A. The formation of this “pre-initiation complex” requires, among other factors, the concerted action of initiation factor eIF2, responsible for binding to the initiator tRNA-Meti, and eIF3, which interacts with the 40S ribosomal subunit (Hershey & Menick. Translational Control of Gene Expression, pp. 33-88, Cold Spring Harbor Laboratory Press, NY 2000).
In the alternative mechanism, translation initiation occurs internally on the transcript and is mediated by an internal ribosome entry sequence (IRES) element that recruits the translational machinery to an internal initiation codon in the mRNA with the help of trans-acting factors (Jackson. Translational Control of Gene Expression, pp. 127-184, Cold Spring Harbor Laboratory Press, NY 2000). IRES elements have been found in numerous transcripts from viruses that infect vertebrate, invertebrate, or plant cells, as well as in transcripts from vertebrate and invertebrate genes.
During many viral infections, as well as in other cellular stress conditions, changes in the phosphorylation state of eIF2, which lower the levels of the ternary complex eIF2-GTP-tRNA-Meti, result in overall inhibition of protein synthesis. Conversely, specific shut-off of cap-dependent initiation depends upon modification of eIF4F functionality (Thompson & Sarnow. Currentt Opinion in Microbiology 3: 366-370 (2000)).
IRES elements bypass cap-dependent translation inhibition; thus the translation directed by an IRES element is termed “cap-independent.” IRES-driven translation initiation prevails during many viral infections, such as, for example, picornaviral infection (Macejak & Sarnow. Nature 353: 90-94 (1991)). Under these circumstances, cap-dependent initiation is inhibited or severely compromised due to the presence of small amounts of functional eIF4F. This is caused by cleavage or loss of solubility of eIF4G (Gradi et al. Proceedings of the National Academy of Sciences, USA 95: 11089-11094 (1998)); 4E-BP dephosphorylation (Gingras et al. Proceedings of the National Academy of Sciences, USA 93: 5578-5583 (1996)) or poly(A)-binding protein (PABP) cleavage (Joachims et al. Journal of Virology 73: 718-727 (1999)).
Alphavirus vectors that express a nucleic acid of interest (NOI) at varying levels have been described. All of these examples describe modification of the alphavirus non-structural protein genes or of the 26S (subgenomic) promoter to regulate vector replication or transcription from the subgenomic promoter. Examples include mutations in the non-structural protein genes that increase or decrease subgenomic RNA transcription or alter genomic RNA replication, resulting in modified NOI expression. Control of protein expression from an alphavirus vector, at the level of translation of the subgenomic mRNA, has not been described previously.
The present invention provides alphavirus replicon and helper vectors engineered to control the expression of one or more heterologous nucleic acid sequences at the level of protein translation via a cap-independent mechanism under the direction of an IRES element.